title: Restoration of CAPAN-1 cells with functional BRCA2 provides insight into the DNA repair activity of individuals who are heterozygous for BRCA2 mutations.
abstract: Mutations in the BRCA2 gene are associated with inherited, early-onset breast cancer. CAPAN-1 cells have been useful for studying how BRCA2 mutations contribute to malignant transformation. They exhibit loss of heterozygosity (LOH), and the remaining copy of BRCA2 has a 6174delT mutation, which causes a premature C-terminal truncation that removes the domains for DNA repair and the nuclear localization signals. The DNA repair protein RAD51, which interacts with BRCA2, exhibits impaired nuclear translocation in CAPAN-1. It has been speculated that RAD51 may require BRCA2 for nuclear entry and that C-terminally truncated BRCA2 may retain RAD51 in the cytoplasm. This may cause heterozygous individuals to exhibit deficient DNA repair and cell viability comparable to individuals with LOH or biallelic BRCA2 mutations. We simulated a heterozygous condition by using stably transfected CAPAN-1 cells with wild-type BRCA2. Fusion of a nuclear localization signal to RAD51 did not increase its ability to independently enter the nuclei of CAPAN-1 cells. Furthermore, restoration of functional BRCA2 did not significantly improve DNA repair, nor did it reestablish cell viability in CAPAN-1 cells. The results imply that C-terminally truncated BRCA2 hinders RAD51 nuclear translocation, possibly contributing to genetic instabilities in homozygous as well as heterozygous individuals.
authors.affiliation: Department of Pathology, University of Colorado Health Sciences Center, Aurora, CO 80010, USA.
authors.forename: Jeffrey T
authors.initials: JT
authors.lastname: Holt
authors.forename: William P
authors.initials: WP
authors.lastname: Toole
authors.forename: Vedang R
authors.initials: VR
authors.lastname: Patel
authors.forename: HeeYoung
authors.initials: H
authors.lastname: Hwang
authors.forename: Erika T
authors.initials: ET
authors.lastname: Brown
mesh_terms: D021581:Active Transport, Cell Nucleus; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000528:radiation effects; D004249:DNA Damage; D004260:DNA Repair; D024522:Genes, BRCA2; D006367:HeLa Cells; D006801:Humans; D009154:Mutation; D019913:Nuclear Localization Signals; D010190:Pancreatic Neoplasms / Q000235:genetics*; D051135:Rad51 Recombinase / Q000378:metabolism
chemical_list: D019913:Nuclear Localization Signals; C495153:RAD51 protein, human; D051135:Rad51 Recombinase